Anti-interferon gamma antibodies and methods of use thereof

ABSTRACT

The invention relates to fully human antibodies, and fragments thereof, that bind to human interferon gamma (hIFNγ), thereby modulating the interaction between IFNγ and its receptor, IFNγ-R, and/or modulating the biological activities of IFNγ. The invention also relates to the use of such anti-IFNγ antibodies in the prevention or treatment of immune-related disorders and in the amelioration of a symptom associated with an immune-related disorder.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/057,356, filed Oct. 18, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/702,013, filed Feb. 8, 2010, now abandoned,which is a continuation of U.S. patent application Ser. No. 11/342,020,filed Jan. 27, 2016, now issued as U.S. Pat. No. 7,700,098, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.60/648,219, filed Jan. 27, 2005, the contents of each of which is herebyincorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “NOVI009C03USSeqList.txt,” which wascreated on Jun. 20, 2017 and is 58.9 KB in size, are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to fully human anti-interferon gammaantibodies as well as to methods for use thereof.

BACKGROUND OF THE INVENTION

Human interferon gamma (IFNγ, IFN-gamma) is a lymphokine produced byactivated T-lymphocytes and natural killer cells. It manifestsanti-proliferative, antiviral and immunomodulatory activities and bindsto IFNγ-R, a heterodimeric receptor on most primary cells of the immunesystem, and triggers a cascade of events leading to inflammation. Theantiviral and immunomodulatory activity of IFNγ is known to havebeneficial effects in a number of clinical conditions. However, thereare many clinical settings in which IFNγ-activity is known to havedeleterious effects. For example, autoimmune diseases are associatedwith high levels of IFNγ in the blood and diseased tissue fromautoimmune patients. IFNγ-activity has also been linked to such diseasestates as cachexia and septic shock.

Accordingly, there exists a need for therapies that target IFNγactivity.

SUMMARY OF THE INVENTION

The present invention provides fully human monoclonal antibodiesspecifically directed against interferon gamma (IFNγ, also referred toherein as IFN-gamma). Exemplary monoclonal antibodies include NI-0501;AC1.2R3P2_A6 (also referred to herein as “A6”); AC1.2R3P2_B4 (alsoreferred to herein as “B4”); AD1.4R4P1_B9 (also referred to herein as“B9”); AD1.4R4P2_C9 (also referred to herein as “C9”); AC1.4R4P2_C10(also referred to herein as “C10”); AC1.2R3P7_D3 (also referred toherein as “D3”); AD1.2R2P2_D6 (also referred to herein as “D6”);AC1.2R2P2_D8 (also referred to herein as “D8”); AD1.3R3P6_E1 (alsoreferred to herein as “E1”); AD1.3R3P5_F8 (also referred to herein as“F8”); AD1.3R3P6_F9 (also referred to herein as “F9”); AD1.4R4P2_G7(also referred to herein as “G7”); AD1.1R3P3_G9 (also referred to hereinas “G9”); and AD1.3R3P6_G10 (also referred to herein as “G10”) describedherein. Alternatively, the monoclonal antibody is an antibody that bindsto the same epitope as NI-0501; AC1.2R3P2_B4; AD1.4R4P1_B9;AD1.4R4P2_C9; AC1.4R4P2_C10; AC1.2R3P7_D3; AD1.2R2P2_D6; AC1.2R2P2_D8;AD1.3R3P6_E1; AD1.3R3P5_F8; AD1.3R3P6_F9; AD1.4R4P2_G7; AD1.1R3P3_G9; orAD1.3R3P6_G10. The antibodies are respectively referred to herein ashuIFNγ antibodies.

A huIFNγ antibody contains a heavy chain variable having the amino acidsequence of SEQ ID NOS: 2, 12, 20, 28, 36, 42, 51, 58, 63, 68, 75, 81,88, 94, or 103 and a light chain variable having the amino acid sequenceof SEQ ID NOS: 7, 15, 23, 31, 38, 47, 54, 60, 66, 71, 78, 83, 91, 96 or105. Preferably, the three heavy chain complementarity determiningregions (CDRs) include an amino acid sequence at least 90%, 92%, 95%,97% 98%, 99% or more identical a sequence selected from the groupconsisting of SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4);DGSSGWYVPHWF DP (SEQ ID NO:5); DHSSGWYVISGMDV (SEQ ID NO:13);DLTVGGPWYYFDY (SEQ ID NO:21); DGWNALGWLES (SEQ ID NO:29); SNAMS (SEQ IDNO:43); TLTGSGGTAYYADSVEG (SEQ ID NO:44); GTELVGGGLDN (SEQ ID NO:45);RSFDSGGSFEY (SEQ ID NO:64); VGSWYLEDFDI (SEQ ID NO:69); GGNYGDYFDYFDY(SEQ ID NO:76); and DFWVITSGNDY (SEQ ID NO:89); and a light chain withthree CDR that include an amino acid sequence at least 90%, 92%, 95%,97% 98%, 99% or more identical to a sequence selected from the groupconsisting of the amino acid sequence of TRSSGSIASNYVQ (SEQ ID NO:8);EDNQRPS (SEQ ID NO:9); QSYDGSNRWM (SEQ ID NO:10); TRSSGSIASNYVQ (SEQ IDNO:16); EDNQRPS (SEQ ID NO:17); QSNDSDNVV (SEQ ID NO:18); DDDQRPS (SEQID NO:25); QSYDSSNVV (SEQ ID NO:26); TRSGGSIGSYYVQ (SEQ ID NO:32);DDKKRPS (SEQ ID NO:33); QSYDSNNLVV (SEQ ID NO:34); TRSSGTIASNYVQ (SEQ IDNO:39); QSYDNSNHWV (SEQ ID NO:40); TGSGGSIATNYVQ (SEQ ID NO:48);QSYDSDNHHVV (SEQ ID NO:49); TGSSGSIASNYVQ (SEQ ID NO:55); QSYDSSNQEVV(SEQ ID NO:56); QSYDSNNFWV (SEQ ID NO:61); TRSSGSIASNYVH (SEQ ID NO:72);QSSDTTYHGGVV (SEQ ID NO:73); QSYEGF (SEQ ID NO:79); TGRNGNIASNYVQ (SEQID NO:84); EDTQRPS (SEQ ID NO:85); QSSDSNRVL (SEQ ID NO:86); QSFDSTNLVV(SEQ ID NO:92); AGSSGSIASNYVQ (SEQ ID NO:97); QSYSYNNQVV (SEQ ID NO:98);TRSSGSIVSNYVQ (SEQ ID NO:106); EDNRRPS (SEQ ID NO:107). The antibodybinds IFNγ.

The huIFNγ antibodies of the invention include a V_(H) CDR1 regioncomprising the amino acid sequence SYAMS (SEQ ID NO:3) or SNAMS (SEQ IDNO:43); a V_(H) CDR2 region comprising the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO:4) or TLTGSGGTAYYADSVEG (SEQ ID NO:44), anda V_(H) CDR3 region comprising an amino acid sequence selected from thegroup consisting of DGSSGWYVPHWFDP (SEQ ID NO:5); DHSSGWYVISGMDV (SEQ IDNO:13); DLTVGGPWYYFDY (SEQ ID NO:21); DGWNALGWLES (SEQ ID NO:29);GTELVGGGLDN (SEQ ID NO:45); RSFDSGGSFEY (SEQ ID NO:64); VGSWYLEDFDI (SEQID NO:69); GGNYGDYFDYFDY (SEQ ID NO:76); and DFWVITSGNDY (SEQ ID NO:89).

The huIFNγ antibodies include a V_(L) CDR1 region comprising an aminoacid sequence selected from the group consisting of TRSSGSIASNYVQ (SEQID NO:8); TRSSGSIASNYVQ (SEQ ID NO:16); TRSGGSIGSYYVQ (SEQ ID NO:32);TRSSGTIASNYVQ (SEQ ID NO:39); TGSGGSIATNYVQ (SEQ ID NO:48);TGSSGSIASNYVQ (SEQ ID NO:55); TRSSGSIASNYVH (SEQ ID NO:72);TGRNGNIASNYVQ (SEQ ID NO:84); AGSSGSIASNYVQ (SEQ ID NO:97) andTRSSGSIVSNYVQ (SEQ ID NO:106); a V_(L) CDR2 region comprising an aminoacid sequence selected from the group consisting of EDNQRPS (SEQ IDNO:9); EDNQRPS (SEQ ID NO:17); DDDQRPS (SEQ ID NO:25); DDKKRPS (SEQ IDNO:33); EDTQRPS (SEQ ID NO:85) and EDNRRPS (SEQ ID NO:107); and a V_(L)CDR3 region comprising an amino acid sequence selected from the groupconsisting of QSYDGSNRWM (SEQ ID NO:10); QSNDSDNVV (SEQ ID NO:18);QSYDSSNVV (SEQ ID NO:26); QSYDSNNLVV (SEQ ID NO:34); QSYDNSNHWV (SEQ IDNO:40); QSYDSDNHHVV (SEQ ID NO:49); QSYDSSNQEVV (SEQ ID NO:56);QSYDSNNFWV (SEQ ID NO:61); QSSDTTYHGGVV (SEQ ID NO:73); QSYEGF (SEQ IDNO:79); QSSDSNRVL (SEQ ID NO:86); QSFDSTNLVV (SEQ ID NO:92); andQSYSYNNQVV (SEQ ID NO:98).

The huIFNγ antibodies include, for example, a V_(H) CDR1 regioncomprising the amino acid sequence SYAMS (SEQ ID NO:3) or SNAMS (SEQ IDNO:43); a V_(H) CDR2 region comprising the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO:4) or TLTGSGGTAYYADSVEG (SEQ ID NO:44); aV_(H) CDR3 region comprising an amino acid sequence selected from thegroup consisting of DGSSGWYVPHWFDP (SEQ ID NO:5); DHSSGWYVISGMDV (SEQ IDNO:13); DLTVGGPWYYFDY (SEQ ID NO:21); DGWNALGWLES (SEQ ID NO:29);GTELVGGGLDN (SEQ ID NO:45); RSFDSGGSFEY (SEQ ID NO:64); VGSWYLEDFDI (SEQID NO:69); GGNYGDYFDYFDY (SEQ ID NO:76); and DFWVITSGNDY (SEQ ID NO:89);a V_(L) CDR1 region comprising an amino acid sequence selected from thegroup consisting of TRSSGSIASNYVQ (SEQ ID NO:8); TRSSGSIASNYVQ (SEQ IDNO:16); TRSGGSIGSYYVQ (SEQ ID NO:32); TRSSGTIASNYVQ (SEQ ID NO:39);TGSGGSIATNYVQ (SEQ ID NO:48); TGSSGSIASNYVQ (SEQ ID NO:55);TRSSGSIASNYVH (SEQ ID NO:72); TGRNGNIASNYVQ (SEQ ID NO:84);AGSSGSIASNYVQ (SEQ ID NO:97) and TRSSGSIVSNYVQ (SEQ ID NO:106); a V_(L)CDR2 region comprising an amino acid sequence selected from the groupconsisting of EDNQRPS (SEQ ID NO:9); EDNQRPS (SEQ ID NO:17); DDDQRPS(SEQ ID NO:25); DDKKRPS (SEQ ID NO:33); EDTQRPS (SEQ ID NO:85) andEDNRRPS (SEQ ID NO:107); and a V_(L) CDR3 region comprising an aminoacid sequence selected from the group consisting of QSYDGSNRWM (SEQ IDNO:10); QSNDSDNVV (SEQ ID NO:18); QSYDSSNVV (SEQ ID NO:26); QSYDSNNLVV(SEQ ID NO:34); QSYDNSNHWV (SEQ ID NO:40); QSYDSDNHHVV (SEQ ID NO:49);QSYDSSNQEVV (SEQ ID NO:56); QSYDSNNFWV (SEQ ID NO:61); QSSDTTYHGGVV (SEQID NO:73); QSYEGF (SEQ ID NO:79); QSSDSNRVL (SEQ ID NO:86); QSFDSTNLVV(SEQ ID NO:92); and QSYSYNNQVV (SEQ ID NO:98).

The heavy chain of a huIFNγ antibody is derived from a germ line V(variable) gene such as, for example, the DP47 (IGHV3-23) germline gene(GenBank Accession No. M99660) or a nucleic acid sequence homologous tothe human DP47 germline gene sequence. The nucleic acid sequence for theDP47 (IGHV 3-23) germline gene includes, for example, the nucleic acidsequence shown below:

(SEQ ID NO: 99) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGA

The light chain of a huIFNγ antibody is derived from a Ig lambda lightchain variable region germline gene such as, for example, the IGLV6-57or V1-22 (GenBank Accession No. Z73673) or a nucleic acid sequencehomologous to the human IGLV6-57 germline gene sequence. The nucleicacid sequence for the IGLV6-57 germline gene includes, for example, thenucleic acid sequence shown below:

(SEQ ID NO: 108) AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAATCA

In another aspect, the invention provides methods of treating,preventing or alleviating a symptom of an immune-related disorder byadministering a huIFNγ antibody to a subject. For example, the huIFNγantibodies are used to treat, prevent or alleviate a symptom associatedwith immune-related disorders such as Crohn's Disease, systemic lupuserythematosus, psoriasis, sarcoidosis, rheumatoid arthritis, vasculitis,atopic dermatitis and secondary progressive multiple sclerosis.Optionally, the subject is further administered with a second agent suchas, but not limited to, an anti-cytokine or anti-chemokine reagent thatrecognizes cytokines such as interleukin 1 (IL-1), IL-2, IL-4, IL-6,IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27 andIL-31, and/or chemokines such as MIP1 alpha, MIP1 beta, RANTES, MCP1,IP-10, ITAC, MIG, SDF and fractalkine.

The subject is suffering from or is predisposed to developing an immunerelated disorder, such as, for example, an autoimmune disease or aninflammatory disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H are a series of representationsof the nucleotide sequence and amino acid sequences for the variablelight and variable heavy regions of the huIFNγ antibodies NI-0501 andAC1.2R3P2_A6. FIG. 1A depicts the nucleotide sequence encoding thevariable region of the heavy chain of NI-0501, and FIG. 1B representsthe amino acid sequence encoded by the nucleotide sequence shown in FIG.1A. The complementarity determining regions (CDRs) are underlined inFIG. 1B. FIG. 1C depicts the nucleotide sequence encoding the variableregion of the light chain of NI-0501, and FIG. 1D represents the aminoacid sequence encoded by the nucleotide sequence shown in FIG. 1C. TheCDRs are underlined in FIG. 1D. FIG. 1E depicts the nucleotide sequenceencoding the variable region of the heavy chain of AC1.2R3P2_A6, andFIG. 1F represents the amino acid sequence encoded by the nucleotidesequence shown in FIG. 1E. The CDRs are underlined in FIG. 1F. FIG. 1Gdepicts the nucleotide sequence encoding the variable region of thelight chain of AC1.2R3P2_A6, and FIG. 1H represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 1G. The CDRsare underlined in FIG. 1H.

FIGS. 2A, 2B, 2C, and 2D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AC1.2R3P2_B4. FIG. 2Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 2B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 2A. The CDRs are underlined inFIG. 2B. FIG. 2C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 2D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 2C. The CDRsare underlined in FIG. 2D.

FIGS. 3A, 3B, 3C, and 3D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.4R4P1_B9. FIG. 3Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 3B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 3A. The CDRs are underlined inFIG. 3B. FIG. 3C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 3D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 3C. The CDRsare underlined in FIG. 3D.

FIGS. 4A, 4B, 4C, and 4D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.4R4P2_C9. FIG. 4Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 4B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 4A. The CDRs are underlined inFIG. 4B. FIG. 4C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 4D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 4C. The CDRsare underlined in FIG. 4D.

FIGS. 5A, 5B, 5C, and 5D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AC1.4R4P2_C10. FIG. 5Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 5B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 5A. The CDRs are underlined inFIG. 5B. FIG. 5C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 5D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 5C. The CDRsare underlined in FIG. 5D.

FIGS. 6A, 6B, 6C, and 6D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AC1.2R3P7_D3. FIG. 6Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 6B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 6A. The CDRs are underlined inFIG. 6B. FIG. 6C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 6D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 6C. The CDRsare underlined in FIG. 6D.

FIGS. 7A, 7B, 7C, and 7D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.2R2P2_D6. FIG. 7Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 7B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 7A. The CDRs are underlined inFIG. 7B. FIG. 7C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 7D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 7C. The CDRsare underlined in FIG. 7D.

FIGS. 8A, 8B, 8C, and 8D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AC1.2R2P2_D8. FIG. 8Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 8B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 8A. The CDRs are underlined inFIG. 8B. FIG. 8C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 8D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 8C. The CDRsare underlined in FIG. 8D.

FIGS. 9A, 9B, 9C, and 9D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.3R3P6_E1. FIG. 9Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 9B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 9A. The CDRs are underlined inFIG. 9B. FIG. 9C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 9D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 9C. The CDRsare underlined in FIG. 9D.

FIGS. 10A, 10B, 10C, and 10D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.3R3P5_F8. FIG. 10Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 10B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 10A. The CDRs are underlined inFIG. 10B. FIG. 10C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 10D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 10C. The CDRsare underlined in FIG. 10D.

FIGS. 11A, 11B, 11C, and 11D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.3R3P6_F9. FIG. 11Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 11B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 11A. The CDRs are underlined inFIG. 11B. FIG. 11C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 11D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 11C. The CDRsare underlined in FIG. 11D.

FIGS. 12A, 12B, 12C, and 12D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.4R4P2_G7. FIG. 12Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 12B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 12A. The CDRs are underlined inFIG. 12B. FIG. 12C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 12D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 12C. The CDRsare underlined in FIG. 12D.

FIGS. 13A, 13B, 13C, and 13D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.1R3P3_G9. FIG. 13Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 13B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 13A. The CDRs are underlined inFIG. 13B. FIG. 13C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 13D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 13C. The CDRsare underlined in FIG. 13D.

FIGS. 14A, 14B, 14C, and 14D are a series of representations of thenucleotide sequence and amino acid sequences for the variable light andvariable heavy regions of the huIFNγ antibody AD1.3R3P6_G10. FIG. 14Adepicts the nucleotide sequence encoding the variable region of theheavy chain, and FIG. 14B represents the amino acid sequence encoded bythe nucleotide sequence shown in FIG. 14A. The CDRs are underlined inFIG. 14B. FIG. 14C depicts the nucleotide sequence encoding the variableregion of the light chain, and FIG. 14D represents the amino acidsequence encoded by the nucleotide sequence shown in FIG. 14C. The CDRsare underlined in FIG. 14D.

FIG. 15 is a graph depicting the inhibition of IFNγ-induced reportergene expression using periplasmic scFv extracts. Quantified scFvextracts inhibited the IFNγ-induced reporter gene in a dose dependentfashion. For each scFv clone various concentrations (2.7, 0.68, 0.17,0.043 and 0.011 nM) were tested as shown by the columns above each clonename (descending concentration from left to right, see also Table 3below).

FIG. 16, Panels 1-12 are a series of graphs depicting the inhibition ofIFNγ-induced MHC class II expression on melanoma cells using scFvextracts. Purified fully human scFv inhibited IFNγ-induced MHC IIexpression on melanoma cells. scFv clones (-) and the mouse anti-humanIFNγ mAb 16C3 (---) are depicted.

FIG. 17, Panels 1-7 are a series of graphs depicting the inhibition ofIFNγ-induced MHC class II expression on melanoma cells using scFvextracts that were reformatted onto a fully human IgG backbone. Purifiedfully IgG mAbs inhibited IFNγ-induced MHC II expression on melanomacells. Fully IgG clones (-x-), the mouse anti-human IFNγ mAb 16C3 (-▴-)and the R&D Systems, Inc. (Minneapolis, Minn.) mouse anti-human IFNγMAB285 (--) are depicted.

FIG. 18 is a graph depicting the affinity of the NI-0501 huIFNγ antibodyfor human IFNγ.

FIG. 19 is a graph comparing the activity of antibodies produced by theA6 and NI-0501 (also referred to herein as “A6 back-mutated to germline”or “back-mutated A6”) clones.

FIG. 20 is a graph depicting the activity of the NI-0501 huIFNγ antibodyon native human IFNγ.

FIGS. 21A-21F are a series of graphs depicting the binding of theNI-0501 huIFNγ antibody with recombinant IFNγ from various species.

FIG. 22 is a graph depicting the ability of the NI-0501 huIFNγ antibodyto neutralize the MHC class II upregulation induced by native cynomolgusIFNγ.

FIG. 23 is a graph depicting the ability of the NI-0501 huIFNγ antibodyto block IFNγ-induced IP-10 production in whole blood.

DETAILED DESCRIPTION

The present invention provides fully human monoclonal antibodiesspecific against interferon gamma (IFNγ). The antibodies arecollectively referred to herein is huIFNγ antibodies.

The huIFNγ antibodies are, for example, IFNγ antagonists or inhibitorsthat modulate at least one biological activity of IFNγ. Biologicalactivities of IFNγ include, for example, binding the IFNγ receptor(IFNγ-R), modulating, e.g., reducing or inhibiting, majorhistocompatibility complex (MHC) class II expression on a cell surface,and modulating, e.g., reducing or inhibiting, cell proliferation. Forexample, the huIFNγ antibodies completely or partially inhibit IFNγactivity by partially or completely blocking the binding of IFNγ and theIFNγ receptor (IFNγ-R). The IFNγ antibodies are considered to completelyinhibit IFNγ activity when the level of IFNγ activity in the presence ofthe huIFNγ antibody is decreased by at least 95%, e.g., by 96%, 97%,98%, 99% or 100% as compared to the level of IFNγ activity in theabsence of binding with a huIFNγ antibody described herein. The IFNγantibodies are considered to partially inhibit IFNγ activity when thelevel of IFNγ activity in the presence of the huIFNγ antibody isdecreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%,75%, 80%, 85% or 90% as compared to the level of IFNγ activity in theabsence of binding with a huIFNγ antibody described herein.

Additionally, the huIFNγ antibodies of the invention inhibitIFNγ-induced MHC class II expression on cells (see e.g., Examples 4 and5). Preferably, the huIFNγ antibodies exhibit greater than 50%inhibition of IFNγ-induced MHC class II expression in the human melanomacell line Me67.8 at a concentration of at least 0.02 nM. For example,the antibodies exhibit greater than 50% inhibition of IFNγ-induced MHCclass II expression in the Me67.8 cell line at a concentration in therange of 0.022 nM to 0.044 nM, e.g., at a concentration of 0.022 nM,0.028 nM or 0.044 nM.

The huIFNγ antibodies modulate an immune response in a subject, e.g., ina human subject. Preferably, the huIFNγ antibodies modulate an adaptiveimmune response in a subject. More preferably, the huIFNγ antibodiesmodulate the cellular or cell-mediated immune response, also known asTh1-type or Th1-mediated response.

For example, the huIFNγ antibodies described herein modulate, e.g.,reduce, inhibit or prevent an exaggerated Th1-mediated immune response,such as an exaggerated Th1-mediated immune response associated with anautoimmune or inflammatory disorder such as, for example, Crohn'sdisease, system lupus erythematosus, psoriasis, sarcoidosis, rheumatoidarthritis, vasculitis, atopic dermatitis and secondary progressivemultiple sclerosis. As used herein, the term “exaggerated” Th1-mediatedimmune response refers to the presence of an elevated level of Th1cytokine(s), such as IL-2, IL-3, TNF-alpha (TNFα) and IFNγ, in a subjectas compared to the level of Th1 cytokine production in a subject notsuffering from a disease or disorder associated with an exaggerated Th1immune response. To classify a Th1-mediated immune response as anexaggerated response, the level of a Th1 cytokine production response isevaluated, e.g., by measuring and analyzing the level of secretedcytokines using an ELISA or other assay.

The huIFNγ antibodies described herein modulate, e.g., inhibit, reduceor prevent, class switching to an IgG isotype, such as IFNγ-inducedclass switching. These huIFNγ antibodies modulate, e.g., inhibit,prevent or reduce a Th1-mediated response and consequently decreaseIFNγ-induced switching.

The huIFNγ antibodies of the invention were produced by immunizing ananimal with IFNγ, such as, for example, murine or human IFNγ (see e.g.,Genbank Accession No. X13274) or an immunogenic fragment, derivative orvariant thereof. Alternatively, the animal is immunized with cellstransfected with a vector containing a nucleic acid molecule encodingIFNγ, such that IFNγ is expressed and associated with the surface of thetransfected cells. Alternatively, the antibodies are obtained byscreening a library that contains antibody or antigen binding domainsequences for binding to IFNγ. This library is prepared, e.g., inbacteriophage as protein or peptide fusions to a bacteriophage coatprotein that is expressed on the surface of assembled phage particlesand the encoding DNA sequences contained within the phage particles(i.e., “phage displayed library”).

huIFNγ antibodies of the invention include, for example, the heavy chaincomplementarity determining regions (CDRs) shown below in Table 1, thelight chain CDRs shown in Table 2, and combinations thereof.

TABLE 1 VH sequences from antibody clones that bind and neutralize IFNγClone Name VH CDR1 VH CDR2 VH CDR3  NI-0501 SYANS AISGSGGSTYYADSVKGDGSSGWYVPHWFDP (SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) AC1.2R3P2_A6SYANS AISGSGGSTYYADSVKG DGSSGWYVPHWFDP (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 5) AC1.2R3P2_D8 SYANS AISGSGGSTYYADSVKG DGSSGWYVPHWFDP(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 5) AC1.4R4P2_C10 SYANSAISGSGGSTYYADSVKG DGSSGWYVPHWFDP (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 5) AC1R3P2_B4 SYANS AISGSGGSTYYADSVKG DHSSGWYVISGMDV(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 13) AD14R4P1_B9 SYANSAISGSGGSTYYADSVKG DLTVGGPWYYFDY (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 21) AD13R3P5_F8 SYANS AISGSGGSTYYADSVKG VGSVYLEDFDI(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 69) AD13RP6_F9 SYANSAISGSGGSTYYADSVKG GGNYGDYFDYFDY (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 76) AD13R3P6_E1 SYANS AISGSGGSTYYADSVKG RSFDSGGSPEY(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 64) AD14R4P2_C9 SYANSAISGSGGSTYYADSVKG DGWNALGWLES (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 29) AD14R4P2_G7 SYANS AISGSGGSTYYADSVKG DGWNALGWLES(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 29) AD13R3P6_G10 SYANSAISGSGGSTYYADSVKG DGWNALGWLES (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 29) AD1R2P2_D6 SYANS AISGSGGSTYYADSVKG DGWNALGWLES(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ ID NO: 29) AD11R3P3_G9 SYANSAISGSGGSTYYADSVKG DVWVITSGNDY (SEQ ID NO: 3) (SEQ ID NO: 4)(SEQ ID NO: 89) AC12R3P7_D3 SNANS TLTGSGGTAYYADSVEG GTELVGGGLDN(SEQ ID NO: 43) (SEQ ID NO: 44) (SEQ ID NO: 45)

TABLE 2 VL sequences from antibody clones that bind and neutralize IFNγClone Name VL CDR1 VL CDR2 VL CDR3  NI-0501 TRSSGSIASNYVQ EDNQRPSQSYDGSNRWM (SEQ ID NO: 8) (SEQ ID NO: 9) (SEQ ID NO: 10) AC1.2R3P2_A6TRSSGSIVSNYVQ EDNRRPS QSYDGSNRWM (SEQ ID NO: 106) (SEQ ID NO: 107)(SEQ ID NO: 10) AC1.2R3P2_D8 TRSSGSIASNYVQ EDNQRPS QSYDSNNFWV(SEQ ID NO: 8) (SEQ ID NO: 17) (SEQ ID NO: 61) AC1.4R4P2_C10TRSSGTIASNYVQ EDNQRPS QSYDNSNHWV (SEQ ID NO: 39) (SEQ ID NO: 17)(SEQ ID NO: 40) AC1R3P2_B4 TRSSGSIASNYVQ EDNQRPS QSNDSDNVV(SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) AD14R4P1_B9TRSSGSIASNYVQ DDDQRPS QSYDSSNVV (SEQ ID NO: 8) (SEQ ID NO: 25)(SEQ ID NO: 26) AD13R3P5_F8 TRSSGSIASNYVH EDNQRPS QSSDTTYHGGVV(SEQ ID NO: 72) (SEQ ID NO: 9) (SEQ ID NO: 73) AD13RP6_F9 TRSSGSIASNYVQEDNQRPS QSYEGF (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 79)AD13R3P6_E1 TRSSGSIASNYVQ EDDRRPS QSYDDTTPWV (SEQ ID NO: 8)(SEQ ID NO: 25) (SEQ ID NO: 26) AD14R4P2_C9 TRSGGSIGSYYVQ DDKKRPSQSYDSNNLVV (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) AD14R4P2_G7TGRNGNIASNYVQ EDTQRPS QSSDSNRVL (SEQ ID NO: 84) (SEQ ID NO: 85)(SEQ ID NO: 86) AD13R3P6_G10 AGSSGSIASNYVQ EDNQRPS QSYSYNNQVV(SEQ ID NO: 97) (SEQ ID NO: 17) (SEQ ID NO: 98) AD1R2P2_D6 TGSSGSIASNYVQEDNQRPS QSYDSSNQEVV (SEQ ID NO: 55) (SEQ ID NO: 17) (SEQ ID NO: 56)AD11R3P3_G9 TRSSGSIASNYVQ EDNQRPS QSFDSTNLVV (SEQ ID NO: 16)(SEQ ID NO: 9) (SEQ ID NO: 92) AC12R3P7_D3 TGSGGSIATNYVQ EDNQRPSQSYDSDNHHVV (SEQ ID NO: 48) (SEQ ID NO: 17) (SEQ ID NO: 49)

An exemplary huIFNγ monoclonal antibody is the NI-0501 antibodydescribed herein. The NI-0501 antibody is a back-mutated version of theAC1.2R3.P2_A6 antibody. As used herein, the term “back-mutated” refersto mutating a nucleotide or amino acid residue back to the nucleotide orresidue found at the corresponding location in the germline sequence.The NI-0501 antibody includes a heavy chain variable region (SEQ IDNO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1, and alight chain variable region (SEQ ID NO:7) encoded by the nucleic acidsequence shown in SEQ ID NO:6 (FIGS. 1A-1D).

The amino acids encompassing the complementarity determining regions(CDR) as defined by Chothia et al. and E. A. Kabat et al. are underlinein FIGS. 1B and 1D. (See Chothia, C, et al., Nature 342:877-883 (1989);Kabat, E A, et al., Sequences of Protein of immunological interest,Fifth Edition, US Department of Health and Human Services, US GovernmentPrinting Office (1991)). The heavy chain CDRs of the A6 antibody havethe following sequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ IDNO:4); and DGSSGWYVPHWFDP (SEQ ID NO:5). The light chain CDRs of the A6antibody have the following sequences: TRSSGSIASNYVQ (SEQ ID NO:8);EDNQRPS (SEQ ID NO:9); and QSYDGSNRWM (SEQ ID NO:10).

Another exemplary huIFNγ monoclonal antibody is the AC1.2R3.P2_A6antibody (“A6”) described herein. The A6 antibody includes a heavy chainvariable region (SEQ ID NO:103) encoded by the nucleic acid sequenceshown in SEQ ID NO:102, and a light chain variable region (SEQ IDNO:105) encoded by the nucleic acid sequence shown in SEQ ID NO:104(FIGS. 1E-1H). The amino acids encompassing the complementaritydetermining regions (CDR) as defined by Chothia et al. and E. A. Kabatet al. are underline in FIGS. 1F and 1H. (See Chothia, C, et al., Nature342:877-883 (1989); Kabat, E A, et al., Sequences of Protein ofimmunological interest, Fifth Edition, US Department of Health and HumanServices, US Government Printing Office (1991)). The heavy chain CDRs ofthe A6 antibody have the following sequences: SYAMS (SEQ ID NO:3);AISGSGGSTYYADSVKG (SEQ ID NO:4); and DGSSGWYVPHWFDP (SEQ ID NO:5). Thelight chain CDRs of the A6 antibody have the following sequences:TRSSGSIVSNYVQ (SEQ ID NO:106); EDNRRPS (SEQ ID NO:107); and QSYDGSNRWM(SEQ ID NO:10).

The AC1.2R3P2_B4 antibody (also referred to herein as “B4”) includes aheavy chain variable region (SEQ ID NO:12) encoded by the nucleic acidsequence shown in SEQ ID NO:11, and a light chain variable region (SEQID NO:15) encoded by the nucleic acid sequence shown in SEQ ID NO:14(FIGS. 2A-2D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 2Band 2D. The heavy chain CDRs of the B4 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDHSSGWYVISGMDV (SEQ ID NO:13). The light chain CDRs of the B4 antibodyhave the following sequences: TRSSGSIASNYVQ (SEQ ID NO:16); EDNQRPS (SEQID NO:17); and QSNDSDNVV (SEQ ID NO:18).

The AD1.4R4P1_B9 antibody (also referred to herein as “B9”) includes aheavy chain variable region (SEQ ID NO:20) encoded by the nucleic acidsequence shown in SEQ ID NO:19, and a light chain variable region (SEQID NO:23) encoded by the nucleic acid sequence shown in SEQ ID NO:22(FIGS. 3A-3D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 3Band 3D. The heavy chain CDRs of the B9 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDLTVGGPWYYFDY (SEQ ID NO:21). The light chain CDRs of the B9 antibodyhave the following sequences: TRSSGSIVSNYVQ (SEQ ID NO:8); DDDQRPS (SEQID NO:25); and QSYDSSNVV (SEQ ID NO:26).

The AD1.4R4P2_C9 antibody (also referred to herein as “C9”) includes aheavy chain variable region (SEQ ID NO:28) encoded by the nucleic acidsequence shown in SEQ ID NO:27, and a light chain variable region (SEQID NO:31) encoded by the nucleic acid sequence shown in SEQ ID NO:30(FIGS. 4A-4D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 4Band 4D. The heavy chain CDRs of the C9 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGWNALGWLES (SEQ ID NO:29). The light chain CDRs of the C9 antibody havethe following sequences: TRSGGSIGSYYVQ (SEQ ID NO:32); DDKKRPS (SEQ IDNO:33); and QSYDSNNLVV (SEQ ID NO:34).

The AC1.4R4P2_C10 antibody (also referred to herein as “C10”) includes aheavy chain variable region (SEQ ID NO:36) encoded by the nucleic acidsequence shown in SEQ ID NO:35, and a light chain variable region (SEQID NO:38) encoded by the nucleic acid sequence shown in SEQ ID NO:37(FIGS. 5A-5D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 5Band 5D. The heavy chain CDRs of the C10 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGSSGWYVPHWF DP (SEQ ID NO:5). The light chain CDRs of the C10 antibodyhave the following sequences: TRSSGTIASNYVQ (SEQ ID NO:39); EDNQRPS (SEQID NO:17); and QSYDNSNHWV (SEQ ID NO:40).

The AC1.2R3P7_D3 antibody (also referred to herein as “D3”) includes aheavy chain variable region (SEQ ID NO:42) encoded by the nucleic acidsequence shown in SEQ ID NO:41, and a light chain variable region (SEQID NO:47) encoded by the nucleic acid sequence shown in SEQ ID NO:46(FIGS. 6A-6D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 6Band 6D. The heavy chain CDRs of the D3 antibody have the followingsequences: SNAMS (SEQ ID NO:43); TLTGSGGTAYYADSVEG (SEQ ID NO:44); andGTELVGGGLDN (SEQ ID NO:45). The light chain CDRs of the D3 antibody havethe following sequences: TGSGGSIATNYVQ (SEQ ID NO:48); EDNQRPS (SEQ IDNO:17) and QSYDSDNHHVV (SEQ ID NO:49).

The AD1.2R2P2_D6 antibody (also referred to herein as “D6”) includes aheavy chain variable region (SEQ ID NO:51) encoded by the nucleic acidsequence shown in SEQ ID NO:50, and a light chain variable region (SEQID NO:54) encoded by the nucleic acid sequence shown in SEQ ID NO:53(FIGS. 7A-7D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 7Band 7D. The heavy chain CDRs of the D6 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGWNALGWLES (SEQ ID NO:29). The light chain CDRs of the D6 antibody havethe following sequences: TGSSGSIASNYVQ (SEQ ID NO:55); EDNQRPS (SEQ IDNO:17); and QSYDSSNQEVV (SEQ ID NO:56).

The AC1.2R2P2_D8 antibody (also referred to herein as “D8”) includes aheavy chain variable region (SEQ ID NO:58) encoded by the nucleic acidsequence shown in SEQ ID NO:57, and a light chain variable region (SEQID NO:60) encoded by the nucleic acid sequence shown in SEQ ID NO:59(FIGS. 8A-8D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 8Band 8D. The heavy chain CDRs of the D8 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGSSGWYVPHWF DP (SEQ ID NO:5). The light chain CDRs of the D8 antibodyhave the following sequences: TRSSGSIVSNYVQ (SEQ ID NO:8); EDNQRPS (SEQID NO:17); and QSYDSNNFWV (SEQ ID NO:61).

The AD1.3R3P6_E1 antibody (also referred to herein as “E1”) includes aheavy chain variable region (SEQ ID NO:63) encoded by the nucleic acidsequence shown in SEQ ID NO:62, and a light chain variable region (SEQID NO:66) encoded by the nucleic acid sequence shown in SEQ ID NO:65(FIGS. 9A-9D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS. 9Band 9D. The heavy chain CDRs of the E1 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andRSFDSGGSFEY (SEQ ID NO:64). The light chain CDRs of the E1 antibody havethe following sequences: TRSSGSIVSNYVQ (SEQ ID NO:8); DDDQRPS (SEQ IDNO:25); and QSYDSSNVV (SEQ ID NO:26).

The AD1.3R3P5_F8 antibody (also referred to herein as “F8”) includes aheavy chain variable region (SEQ ID NO:68) encoded by the nucleic acidsequence shown in SEQ ID NO:67, and a light chain variable region (SEQID NO:71) encoded by the nucleic acid sequence shown in SEQ ID NO:70(FIGS. 10A-10D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS.10B and 10D. The heavy chain CDRs of the F8 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andVGSWYLEDFDI (SEQ ID NO:69). The light chain CDRs of the F8 antibody havethe following sequences: TRSSGSIASNYVH (SEQ ID NO:72); EDNRRPS (SEQ IDNO:9); and QSSDTTYHGGVV (SEQ ID NO:73).

The AD1.3R3P6_F9 antibody (also referred to herein as “F9”) includes aheavy chain variable region (SEQ ID NO:75) encoded by the nucleic acidsequence shown in SEQ ID NO:74, and a light chain variable region (SEQID NO:78) encoded by the nucleic acid sequence shown in SEQ ID NO:77(FIGS. 11A-11D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS.11B and 11D. The heavy chain CDRs of the F9 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andGGNYGDYFDYFDY (SEQ ID NO:76). The light chain CDRs of the F9 antibodyhave the following sequences: TRSSGSIASNYVQ (SEQ ID NO:16); EDNQRPS (SEQID NO:17); and QSYEGF (SEQ ID NO:79).

The AD1.4R4P2_G7 antibody (also referred to herein as “G7”) includes aheavy chain variable region (SEQ ID NO:81) encoded by the nucleic acidsequence shown in SEQ ID NO:80, and a light chain variable region (SEQID NO:83) encoded by the nucleic acid sequence shown in SEQ ID NO:82(FIGS. 12A-12D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS.12B and 12D. The heavy chain CDRs of the G7 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGWNALGWLES (SEQ ID NO:29). The light chain CDRs of the G7 antibody havethe following sequences: TGRNGNIASNYVQ (SEQ ID NO:84); EDTQRPS (SEQ IDNO:85); and QSSDSNRVL (SEQ ID NO:86).

The AD1.1R3P3_G9 antibody (also referred to herein as “G9”) includes aheavy chain variable region (SEQ ID NO:88) encoded by the nucleic acidsequence shown in SEQ ID NO:87, and a light chain variable region (SEQID NO:91) encoded by the nucleic acid sequence shown in SEQ ID NO:90(FIGS. 13A-13D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS.13B and 13D. The heavy chain CDRs of the G9 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDFWVITSGNDY (SEQ ID NO:89). The light chain CDRs of the G9 antibody havethe following sequences: TRSSGSIASNYVQ (SEQ ID NO:16); EDNRRPS (SEQ IDNO:9); and QSFDSTNLVV (SEQ ID NO:92).

The AD1.3R3P6_G10 antibody (also referred to herein as “G10”) includes aheavy chain variable region (SEQ ID NO:94) encoded by the nucleic acidsequence shown in SEQ ID NO:93, and a light chain variable region (SEQID NO:96) encoded by the nucleic acid sequence shown in SEQ ID NO:95(FIGS. 14A-14D). The amino acids encompassing the CDR as defined byChothia et al. 1989, E. A. Kabat et al., 1991 are underlined in FIGS.14B and 14D. The heavy chain CDRs of the G10 antibody have the followingsequences: SYAMS (SEQ ID NO:3); AISGSGGSTYYADSVKG (SEQ ID NO:4); andDGWNALGWLES (SEQ ID NO:29). The light chain CDRs of the G10 antibodyhave the following sequences: AGSSGSIASNYVQ (SEQ ID NO:97); EDNQRPS (SEQID NO:17); and QSYSYNNQVV (SEQ ID NO:98).

huIFNγ antibodies of the invention also include antibodies that includea heavy chain variable amino acid sequence that is at least 90%, 92%,95%, 97% 98%, 99% or more identical the amino acid sequence of SEQ IDNO: 2, 12, 20, 28, 36, 42, 51, 58, 63, 68, 75, 81, 88, 94, or 103 (FIGS.1-14) and/or a light chain variable amino acid that is at least 90%,92%, 95%, 97% 98%, 99% or more identical the amino acid sequence of SEQID NO: 7, 15, 23, 31, 38, 47, 54, 60, 66, 71, 78, 83, 91, 96 or 105(FIGS. 1-14).

Alternatively, the monoclonal antibody is an antibody that binds to thesame epitope as NI-0501, A6, B4, B9, C9, C10, D3, D6, D8, E1, F8, F9,G7, G9 or G10.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. Such antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, single chain, F_(ab),F_(ab′) and F_((ab′)2) fragments, and an F_(ab) expression library. By“specifically bind” or “immunoreacts with” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react (i.e., bind) with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶) with other polypeptides.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y.(1989)). The variable regions of each light/heavy chain pair form theantibody binding site.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, a scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM; preferably ≦100nM and most preferably ≦10 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides are quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to an IFNγ epitope when the equilibriumbinding constant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the invention(e.g., monoclonal antibody NI-0501, A6, B4, B9, C9, C10, D3, D6, D8, E1,F8, F9, G7, G9 or G10) by ascertaining whether the former prevents thelatter from binding to a IFNγ antigen polypeptide. If the humanmonoclonal antibody being tested competes with a human monoclonalantibody of the invention, as shown by a decrease in binding by thehuman monoclonal antibody of the invention, then the two monoclonalantibodies bind to the same, or a closely related, epitope. Another wayto determine whether a human monoclonal antibody has the specificity ofa human monoclonal antibody of the invention is to pre-incubate thehuman monoclonal antibody of the invention with the IFNγ antigenpolypeptide with which it is normally reactive, and then add the humanmonoclonal antibody being tested to determine if the human monoclonalantibody being tested is inhibited in its ability to bind the IFNγantigen polypeptide. If the human monoclonal antibody being tested isinhibited then, in all likelihood, it has the same, or functionallyequivalent, epitopic specificity as the monoclonal antibody of theinvention.

Various procedures known within the art are used for the production ofthe monoclonal antibodies directed against a protein such as an IFNγprotein, or against derivatives, fragments, analogs homologs ororthologs thereof. (See, e.g., Antibodies: A Laboratory Manual, HarlowE, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference). Fully human antibodiesare antibody molecules in which the entire sequence of both the lightchain and the heavy chain, including the CDRs, arise from human genes.Such antibodies are termed “human antibodies”, or “fully humanantibodies” herein. Human monoclonal antibodies are prepared, forexample, using the procedures described in the Examples provided below.Human monoclonal antibodies can be also prepared by using triomatechnique; the human B-cell hybridoma technique (see Kozbor, et al.,1983 Immunol Today 4: 72); and the EBV hybridoma technique to producehuman monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Humanmonoclonal antibodies may be utilized and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

It is desirable to modify the antibody of the invention with respect toeffector function, so as to enhance, e.g., the effectiveness of theantibody in treating immune-related diseases. For example, cysteineresidue(s) can be introduced into the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated can have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)).Alternatively, an antibody can be engineered that has dual Fc regionsand can thereby have enhanced complement lysis and ADCC capabilities.(See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2) huIFNγfragments, single chain huIFNγ antibodies, bispecific huIFNγ antibodiesand heteroconjugate huIFNγ antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for IFNγ. The second binding target is any otherantigen, and advantageously is a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, IFNγ, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII(IFNγ2) and FcγRIII (CD16) so as to focus cellular defense mechanisms tothe cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling is accomplished by any chemical reaction that will bind the twomolecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding is achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987). Preferred linkers are describedin the literature. (See, for example, Ramakrishnan, S. et al., CancerRes. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NETS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules represented by FIGS. 1B,2B, 3B and 4B and the human light chain immunoglobulin moleculesrepresented by FIGS. 1D, 2D, 3D and 4D, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, the lefthanddirection is the amino terminal direction and the righthand direction isthe carboxy-terminal direction, in accordance with standard usage andconvention.

Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5′ end the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long′ morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to IFNγ, under suitablebinding conditions, (2) ability to block appropriate IFNγ binding, or(3) ability to inhibit IFNγ-expressing cell growth in vitro or in vivo.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects. The termsubject includes humans and other mammals.

Human Antibodies and Humanization of Antibodies

A huIFNγ antibody is generated, for example, using the proceduresdescribed in the Examples provided below. An IgG huIFNγ antibody isgenerated, for example, by converting a scFv clone an IgG format (seee.g., Example 6). Alternatively, such a huIFNγ antibody is developed,for example, using phase-display methods using antibodies containingonly human sequences. Such approaches are well-known in the art, e.g.,in WO92/01047 and U.S. Pat. No. 6,521,404, which are hereby incorporatedby reference. In this approach, a combinatorial library of phagecarrying random pairs of light and heavy chains are screened usingnatural or recombinant source of IFNγ or fragments thereof.

A huIFNγ antibody is produced by a process wherein at least one step ofthe process includes immunizing a transgenic, non-human animal withhuman IFNγ protein. Some of the endogenous heavy and/or kappa lightchain loci of this xenogenic non-human animal have been disabled and areincapable of the rearrangement required to generate genes encodingimmunoglobulins in response to an antigen. In addition, at least onehuman heavy chain locus and at least one human light chain locus havebeen stably transfected into the animal. Thus, in response to anadministered antigen, the human loci rearrange to provide genes encodinghuman variable regions immunospecific for the antigen. Uponimmunization, therefore, the xenomouse produces B-cells that secretefully human immunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181and U.S. Pat. No. 6,150,584. By one strategy, the xenogeneic (human)heavy and light chain immunoglobulin genes are introduced into the hostgerm line (e.g., sperm or oocytes) and, in separate steps, thecorresponding host genes are rendered non-functional by inactivationusing homologous recombination. Human heavy and light chainimmunoglobulin genes are reconstructed in an appropriate eukaryotic orprokaryotic microorganism, and the resulting DNA fragments areintroduced into the appropriate host, for example, the pronuclei offertilized mouse oocytes or embryonic stem cells. Inactivation of theendogenous host immunoglobulin loci is achieved by targeted disruptionof the appropriate loci by homologous recombination in the host cells,particularly embryonic stem cells or pronuclei of fertilized mouseoocytes. The targeted disruption can involve introduction of a lesion ordeletion in the target locus, or deletion within the target locusaccompanied by insertion into the locus, e.g., insertion of a selectablemarker. In the case of embryonic stem cells, chimeric animals aregenerated which are derived in part from the modified embryonic stemcells and are capable of transmitting the genetic modifications throughthe germ line. The mating of hosts with introduced human immunoglobulinloci to strains with inactivated endogenous loci will yield animalswhose antibody production is purely xenogeneic, e.g., human.

In an alternative strategy, at least portions of the human heavy andlight chain immunoglobulin loci are used to replace directly thecorresponding endogenous immunoglobulin loci by homologous recombinationin embryonic stem cells. This results in simultaneous inactivation andreplacement of the endogenous immunoglobulin. This is followed by thegeneration of chimeric animals in which the embryonic stem cell-derivedcells can contribute to the germ lines.

For example, a B cell clone that expresses human anti-IFNγ antibody isremoved from the xenogenic non-human animal and immortalized accordingto various methods known within the art. Such B cells may be deriveddirectly from the blood of the animal or from lymphoid tissues,including but not restricted to spleen, tonsils, lymph nodes, and bonemarrow. The resultant, immortalized B cells may be expanded and culturedin vitro to produce large, clinically applicable quantities of huIFNγantibody. Alternatively, genes encoding the immunoglobulins with one ormore human variable regions can be recovered and expressed in adiffering cell type, including but not restricted to a mammalian cellculture system, in order to obtain the antibodies directly or individualchains thereof, composed of single chain F_(v) molecules.

In addition, the entire set of fully human anti-IFNγ antibodiesgenerated by the xenogenic non-human animal may be screened to identifyone such clone with the optimal characteristics. Such characteristicsinclude, for example, binding affinity to the human IFNγ protein,stability of the interaction as well as the isotype of the fully humananti-IFNγ antibody. Clones from the entire set which have the desiredcharacteristics then are used as a source of nucleotide sequencesencoding the desired variable regions, for further manipulation togenerate antibodies with the-se characteristics, in alternative cellsystems, using conventional recombinant or transgenic techniques.

This general strategy was demonstrated in connection with generation ofthe first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). This approach is further discussed anddelineated in U.S. patent application Ser. No. 07/466,008, filed Jan.12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297,filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filedSer. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filedAug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.08/376,279, filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995,Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun.5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837,filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No.08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995,Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct.2, 1996, and Ser. No. 08/759,620, filed Dec. 3, 1996 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoMendez et al. Nature Genetics 15:146-156 (1997) and Green and JakobovitsJ. Exp. Med.: 188:483-495 (1998). See also European Patent No., EP 0 463151 B1, grant published Jun. 12, 1996, International Patent ApplicationNo., WO 94/02602, published Feb. 3, 1994, International PatentApplication No., WO 96/34096, published Oct. 31, 1996, WO 98/24893,published Jun. 11, 1998, WO 00/76310, published Dec. 21, 2000.

In an alternative approach, others have utilized a “minilocus” approach.In the minilocus approach, an exogenous Ig locus is mimicked through theinclusion of pieces (individual genes) from the Ig locus. Thus, one ormore VH genes, one or more DH genes, one or more J_(H) genes, a muconstant region, and a second constant region (preferably a gammaconstant region) are formed into a construct for insertion into ananimal. This approach is described in U.S. Pat. No. 5,545,807 to Suraniet al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877, 397, 5,874,299, and6,255,458 each to Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and6,023,010 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi andDunn, and GenPharm International U.S. patent application Ser. No.07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug. 31,1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No. 07/853,408,filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun. 23, 1992, Ser. No.07/990,860, filed Dec. 16, 1992, Ser. No. 08/053,131, filed Apr. 26,1993, Ser. No. 08/096,762, filed Jul. 22, 1993, Ser. No. 08/155,301,filed Nov. 18, 1993, Ser. No. 08/161,739, filed Dec. 3, 1993, Ser. No.08/165,699, filed Dec. 10, 1993, Ser. No. 08/209,741, filed Mar. 9,1994. See also European Patent No. 0 546 073 B1, International PatentApplication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO98/24884 and U.S. Pat. No. 5,981,175. See further Taylor et al., 1992,Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg etal., (1994), Taylor et al., (1994), and Tuaillon et al., (1995),Fishwild et al., (1996).

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. Commensurately, however, a significantdisadvantage of the minilocus approach is that, in theory, insufficientdiversity is introduced through the inclusion of small numbers of V, D,and J genes. Indeed, the published work appears to support this concern.B-cell development and antibody production of animals produced throughuse of the minilocus approach appear stunted. Therefore, researchsurrounding the present invention has consistently been directed towardsthe introduction of large portions of the Ig locus in order to achievegreater diversity and in an effort to reconstitute the immune repertoireof the animals.

Generation of human antibodies from mice in which, through microcellfusion, large pieces of chromosomes, or entire chromosomes, have beenintroduced, has also been demonstrated. See European Patent ApplicationNos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and an immune variable region,it is expected that certain human anti-chimeric antibody (HACA)responses will be observed, particularly in chronic or multi-doseutilizations of the antibody. Thus, it would be desirable to providefully human antibodies against IFNγ in order to vitiate concerns and/oreffects of HAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization and display techniques using appropriatelibraries. It will be appreciated that murine antibodies or antibodiesfrom other species can be humanized or primatized using techniques wellknown in the art. See e.g., Winter and Harris Immunol Today 14:43 46(1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). Theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (See WO 92102190 and U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). Also, the use of Ig cDNA for construction of chimericimmunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439(1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effecter functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as priers to introduce useful restriction sitesinto the J region for subsequent linkage of V region segments to human Cregion segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL-31sequence can be easily inserted and expressed. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C region, and also atthe splice regions that occur within the human CH exons. Polyadenylationand transcription termination occur at native chromosomal sitesdownstream of the coding regions. The resulting chimeric antibody may bejoined to any strong promoter, including retroviral LTRs, e.g., SV-40early promoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Roussarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloneymurine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also,as will be appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau PEAS USA 94:4937-4942(1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988)(phage display), Scott TIBS 17:241-245 (1992), Cwirla et al. PNAS USA87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085(1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell andMcCafferty TIBTECH; 10:80-8A (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated to IFNγ expressingcells, IFNγ itself, forms of IFNγ, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed above.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto IFNγ, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, generation of peptidetherapeutics, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to IFNγ and another to a second molecule that are conjugatedtogether, (ii) a single antibody that has one chain specific to IFNγ anda second chain specific to a second molecule, or (iii) a single chainantibody that has specificity to IFNγ and the other molecule. Suchbispecific antibodies are generated using techniques that are well knownfor example, in connection with (i) and (ii) See e.g., Fanger et al.Immunol Methods 4:72-81 (1994) and Wright and Harris, supra, and inconnection with (iii) See e.g., Traunecker et al. Int. J. Cancer(Suppl.) 7:51-52 (1992).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing IFNγ, and particularly those cells inwhich the antibodies of the invention are effective.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to IFNγ and antibodiesthereto, such as the antibodies of the invention or screening of peptidelibraries, therapeutic peptides can be generated that are directedagainst IFNγ. Design and screening of peptide therapeutics is discussedin connection with Houghten et al. Biotechniques 13:412-421 (1992),Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al. Biotechniques13:901-905 (1992), Blake and Litzi-Davis BioConjugate Chem. 3:510-513(1992). Immunotoxins and radiolabeled molecules can also be prepared,and in a similar manner, in connection with peptidic moieties asdiscussed above in connection with antibodies. Assuming that the IFNγmolecule (or a form, such as a splice variant or alternate form) isfunctionally active in a disease process, it will also be possible todesign gene and antisense therapeutics thereto through conventionaltechniques. Such modalities can be utilized for modulating the functionof IFNγ. In connection therewith the antibodies of the present inventionfacilitate design and use of functional assays related thereto. A designand strategy for antisense therapeutics is discussed in detail inInternational Patent Application No. WO 94/29444. Design and strategiesfor gene therapy are well known. However, in particular, the use of genetherapeutic techniques involving intrabodies could prove to beparticularly advantageous. See e.g., Chen et al. Human Gene Therapy5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997). General designof and considerations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the IFNγ molecule and itsinteractions with other molecules in accordance with the presentinvention, such as the antibodies of the invention, and others can beutilized to rationally design additional therapeutic modalities. In thisregard, rational drug design techniques such as X-ray crystallography,computer-aided (or assisted) molecular modeling (CAMM), quantitative orqualitative structure-activity relationship (QSAR), and similartechnologies can be utilized to focus drug discovery efforts. Rationaldesign allows prediction of protein or synthetic structures which caninteract with the molecule or specific forms thereof which can be usedto modify or modulate the activity of IFNγ. Such structures can besynthesized chemically or expressed in biological systems. This approachhas been reviewed in Capsey et al. Genetically Engineered HumanTherapeutic Drugs (Stockton Press, NY (1988)). Further, combinatoriallibraries can be designed and synthesized and used in screeningprograms, such as high throughput screening efforts.

Therapeutic Administration and Formulations

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include a huIFNγantibody of the invention, are used to treat or alleviate a symptomassociated with an immune-related disorder, such as, for example, anautoimmune disease or an inflammatory disorder.

For example, administering a huIFNγ antibody to a subject suffering fromCrohn's Disease (CD) can act directly on the disease-causing immunecells, thereby providing rapid intervention with minimal suppression ofthe immune system. Administering a huIFNγ antibody to a subjectsuffering from Systemic Lupus Erythematosus is another medicalindication that provides an opportunity to modify the immune cellsresponsible for the disease. Administering an huIFNγ antibody, a fullyhuman protein, to a subject suffering from psoriasis avoids the need totreat patients with more aggressive medications (e.g., Methotrexate)that have well documented unwanted side effects (e.g., liver damage).Administering a huIFNγ antibody to a subject suffering from rheumatoidarthritis is another medical indication that provides the opportunity tomodulate the upstream generation and function of disease-inducingTh1-mediated response.

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Ménière's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

The huIFNγ antibodies modulate an immune response in a subject, e.g., ina human subject. For example, the huIFNγ antibodies described hereinmodulate, e.g., reduce, inhibit or prevent an exaggerated Th1-mediatedimmune response, such as an exaggerated Th1-mediated immune responseassociated with an autoimmune or inflammatory disorder such as, forexample, Crohn's disease, system lupus erythematosus, psoriasis,sarcoidosis, rheumatoid arthritis, vasculitis, atopic dermatitis andsecondary progressive multiple sclerosis. In an exaggerated Th1-mediatedimmune response, Th1 cytokine(s), such as IL-2, IL-3, TNF-alpha (TNFα)and IFNγ, are presented in a subject at level that is higher than thelevel of Th1 cytokine production in a subject not suffering from adisease or disorder associated with an exaggerated Th-1 immune response.To classify a Th1-mediated immune response as an exaggerated response,the level of a Th1 cytokine production response is evaluated, e.g., bymeasuring and analyzing the level of secreted cytokines using an ELISAor other assay.

The huIFNγ antibodies described herein are also used to modulate, e.g.,inhibit, reduce or prevent, class switching to an IgG isotype, such asIFNγ-induced class switching. These huIFNγ antibodies modulate, e.g.,inhibit, prevent or reduce a Th1-mediated response and consequentlydecrease IFNγ-induced switching.

In one embodiment, the huIFNγ antibody compositions used to treat animmune-related disorder are administered in combination with any of avariety of anti-cytokine agents or anti-chemokine agents. Suitableanti-cytokine or anti-chemokine reagents recognize, for example,cytokines such as interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-12, IL-13,IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27 and IL-31, and/orchemokines such as MIP1 alpha, MIP1 beta, RANTES, MCP1, IP-10, ITAC,MIG, SDF and fractalkine.

The present invention also provides methods of treating or alleviating asymptom associated with an immune-related disorder. For example, thecompositions of the invention are used to treat or alleviate a symptomof any of the autoimmune diseases and inflammatory disorders describedherein. Symptoms associated with immune-related disorders include, forexample, inflammation, fever, loss of appetite, weight loss, abdominalsymptoms such as, for example, abdominal pain, diarrhea or constipation,joint pain or aches (arthralgia), fatigue, rash, anemia, extremesensitivity to cold (Raynaud's phenomenon), muscle weakness, musclefatigue, changes in skin or tissue tone, shortness of breath or otherabnormal breathing patterns, chest pain or constriction of the chestmuscles, abnormal heart rate (e.g., elevated or lowered), lightsensitivity, blurry or otherwise abnormal vision, and reduced organfunction.

The therapeutic formulations of huIFNγ antibody are administered to asubject suffering from an immune-related disorder, such as an autoimmunedisease or an inflammatory disorder. A subject suffering from anautoimmune disease or an inflammatory disorder is identified by methodsknown in the art. For example, subjects suffering from an autoimmunedisease such as Crohn's disease, lupus or psoriasis, are identifiedusing any of a variety of clinical and/or laboratory tests such as,physical examination, radiologic examination and blood, urine and stoolanalysis to evaluate immune status. For example, patients suffering fromlupus are identified, e.g., by using the anti-nuclear antibody test(ANA) to determine if auto-antibodies to cell nuclei are present in theblood. Patients suffering from Crohn's are identified, e.g., using anupper gastrointestinal (GI) series and/or a colonoscopy to evaluate thesmall and large intestines, respectively. Patients suffering frompsoriasis are identified, e.g., using microscopic examination of tissuetaken from the affected skin patch, while patients suffering fromrheumatoid arthritis are identified using, e.g., blood tests and/orx-ray or other imaging evaluation.

Administration of a huIFNγ antibody to a patient suffering from animmune-related disorder such as an autoimmune disease or an inflammatorydisorder if any of a variety of laboratory or clinical results isachieved. For example, administration of a huIFNγ antibody to a patientsuffering from an immune-related disorder such as an autoimmune diseaseor an inflammatory disorder is considered successful one or more of thesymptoms associated with the disorder is alleviated, reduced, inhibitedor does not progress to a further, i.e., worse, state. Administration ofa huIFNγ antibody to a patient suffering from an immune-related disordersuch as an autoimmune disease or an inflammatory disorder is consideredsuccessful if the disorder, e.g., an autoimmune disorder, entersremission or does not progress to a further, i.e., worse, state.

Diagnostic and Prophylactic Formulations

The fully human anti-IFNγ MAbs of the invention are used in diagnosticand prophylactic formulations. In one embodiment, a huIFNγ MAb of theinvention is administered to patients that are at risk of developing oneof the aforementioned autoimmune diseases. A patient's predisposition toone or more of the aforementioned autoimmune diseases can be determinedusing genotypic, serological or biochemical markers.

In another embodiment of the invention, a huIFNγ antibody isadministered to human individuals diagnosed with one or more of theaforementioned autoimmune diseases. Upon diagnosis, a huIFNγ antibody isadministered to mitigate or reverse the effects of autoimmunity.

Antibodies of the invention are also useful in the detection of IFNγ inpatient samples and accordingly are useful as diagnostics. For example,the huIFNγ antibodies of the invention are used in in vitro assays,e.g., ELISA, to detect IFNγ levels in a patient sample.

In one embodiment, a huIFNγ antibody of the invention is immobilized ona solid support (e.g., the well(s) of a microtiter plate). Theimmobilized antibody serves as a capture antibody for any IFNγ that maybe present in a test sample. Prior to contacting the immobilizedantibody with a patient sample, the solid support is rinsed and treatedwith a blocking agent such as mink protein or albumin to preventnonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample is, e.g., a serum sample from a subjectsuspected of having levels of circulating antigen considered to bediagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of IFNγ antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using thehuIFNγ antibodies of the invention in an in vitro diagnostic assay, itis possible to stage a disease (e.g., an autoimmune or inflammatorydisorder) in a subject based on expression levels of the IFNγ antigen.For a given disease, samples of blood are taken from subjects diagnosedas being at various stages in the progression of the disease, and/or atvarious points in the therapeutic treatment of the disease. Using apopulation of samples that provides statistically significant resultsfor each stage of progression or therapy, a range of concentrations ofthe antigen that may be considered characteristic of each stage isdesignated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

Examples

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1: Cloning, Expression and Purification of Human InterferonGamma Cloning.

The sequence corresponding to the mature sequence of human interferongamma (hIFNγ, huIFNγ) was amplified from human cDNA by polymerase chainreaction (PCR) using specific oligonucleotides. The amplificationproduction was gel-purified and cloned into the pET41c expression vector(Novagen, San Diego Calif.). The vector was further modified tointroduce an Avitag™ (Avidity, Denver Colo.) and an octa-histidine tagat the C-terminus of hIFNγ allowing for in vivo biotinylation of theprotein and purification by IMAC (Immobilized Metal Ion AffinityChromatography).

Expression.

E. coli BL21 cells were co-transformed with the pET41c-hIFNγ and apACYC184-BirA vector, which encodes the BirA enzyme required for the invivo biotinylation of the Avitag™ sequence. Single colonies resistant toKanamycin (50 μg/ml) and Chloramphenicol (10 μg/ml) were selected andused to inoculate a starter culture in LB (Kan 50 μg/ml/Cm 10 μg/ml) andgrown overnight at 37° C.

The next day, the culture was used to inoculate (1:100 dilution) a 400ml culture of LB (Kan 50 μg/ml/Cm 10 μg/ml) supplemented with 50 μMbiotin. The culture was grown at 37° C. with shaking (240 rpm) until anOD₆₀₀ of 0.6 was reached. At that point,isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a finalconcentration of 1 mM, and the culture was further incubated for 3 hunder the same conditions. Cells were centrifuged at 4000 rpm for 20minutes, and the pellet was frozen at −20° C. Under these conditionsessentially all of the hIFNγ was insoluble and found in inclusionbodies.

Purification.

Bacterial pellets were thawed and resuspended in 8 ml of Bugbusterreagent containing 8 μl of Benzonaze (Novagen) and incubated at roomtemperature for 30 minutes. The solution was centrifuged for 30 minutesat 15′000 g at 4° C. The pellet containing the inclusion bodies wasresuspended in 7 ml of solubilization buffer (50 mM Tris-HCL pH 7.4, 300mM NaCl, 20 mM Imidazole, 5 mM β-mercaptoethanol, 6M Guanidin-HCl). Theresuspended material was centrifuged at 4° C. for 30 minutes at 15′000g.

Two 5 ml Hi Trap Chelating column (Amersham, Buckinghamshire, England),loaded with NiSO₄ and equilibrated with solubilization buffer, wereconnected together according to manufacturer's instructions. Thesupernatant after the centrifugation step was filter on a 0.45 μmmembrane and loaded on the column with the help of peristaltic pump at 1ml/min. The columns were then placed on an AKTA prime chromatographysystem for on column protein refolding and elution. The immobilizedprotein was washed with 35 ml of solubilization buffer at 1 ml/min. Alinear gradient of solubilization buffer with increasing concentrationof refolding buffer (50 mM Tris-HCL pH 7.4, 300 mM NaCl) was applied at1 ml/min. for 1 hour until 100% refolding buffer was reached. The columnwas then further washed with 25 ml of refolding buffer. The refoldedprotein was then eluted from the column with elution buffer (50 mMTris-HCl, 300 mM NaCl, 400 mM Imidazole). Protein containing fractionswere pooled and desalted on PD10 columns (Amersham) equilibrated withPBS. The desalted protein was then aliquoted and stored at −80° C.

Example 2: Cells Expressing Interferon Gamma on Cell Surface

Chinese hamster ovary (CHO) cells (available from ATCC) were stablytransfected with c-myc-tagged human IFNγ cDNA. cDNAs were subcloned intopCDNA 3.1 plasmids (Invitrogen, Carlsbad Calif.) containing neomycinresistance genes. Transfectants were selected by using this antibiotic,and successive cell sorting was accomplished by flow cytometry using ananti-6xHis (Sigma) antibody. Surface expression of human IFNγ wasconfirmed via flow cytometry using an anti-IFNγ mAb (clone B27, BectonDickinson, Franklin Lakes N.J.).

Example 3: Screening of Human scFv Libraries

General procedures for construction and handling of human scFv librariesare described in Vaughan et al., (Nat. Biotech. 1996, 14:309-314),hereby incorporated by reference in its entirety. Libraries of humanscFv were screened against hIFNγ according to the following procedure.

Liquid Phase Selections.

Aliquots of scFv phage libraries (10¹² Pfu) obtained from CambridgeAntibody Technology (Cambridge, UK) were blocked with PBS containing 3%(w/v) skimmed milk for one hour at room temperature on a rotary mixer.Blocked phage was then deselected on streptavidin magnetic beads (DynalM-280) for one hour at room temperature on a rotary mixer. Deselectedphage was then incubated with in vivo biotinylated hIFNγ (100 nM) fortwo hours at room temperature on a rotary mixer. Beads were capturedusing a magnetic stand followed by four washes with PBS/0.1% Tween 20and 3 washes with PBS. Beads were then directly added to 10 ml ofexponentially growing TG1 cells and incubated for one hour at 37° C.with slow shaking (100 rpm). An aliquot of the infected TG1 was serialdiluted to titer the selection output. The remaining infected TG1 werespun at 3000 rpm for 15 minutes and re-suspended in 0.5 ml 2xTY-AG (2xTYmedia containing 100 μg/ml ampicillin and 2% glucose) and spread on2xTYAG agar Bioassay plates. After overnight incubation at 30° C. 10 mlof 2xTYAG was added to the plates and the cells were scraped form thesurface and transferred to a 50 ml polypropylene tube. 2xTYAG containing50% glycerol was added to the cell suspension to obtain a finalconcentration of 17% glycerol. Aliquots of the selection round were keptat −80° C.

Cell Surface Selections.

Aliquots of scFv phage libraries (10¹² Pfu) obtained from CambridgeAntibody Technology (Cambridge, UK) were blocked with PBS containing 3%(w/v) skimmed milk for one hour at room temperature on a rotary mixer.Blocked phage was then deselected for one hour at 37° C./5% CO₂ on CHOcells transfected with an empty pDisplay vector (in a T75 flask 80%confluence) and that had been previously blocked with PBS containing 2%(w/v) skimmed milk. Deselected phage was then incubatedCHO-pDisplay-hIFNγ cells for one hour at room temperature with gentleshaking. Cells were then washed ten times with PBS. Bound phage waseluted by adding directly 10 ml of exponentially growing TG1 to the T75flask and incubating for one hour at 37° C. with slow shaking. Analiquot of the infected TG1 was serial diluted to titer the selectionoutput. Infected TG1 were spun at 3000 rpm for 15 minutes andre-suspended in 0.5 ml 2xTY-AG (2xTY media containing 100 μg/mlampicillin and 2% glucose) and spread on 2xTYAG agar Bioassay plates.After overnight incubation at 30° C. 10 ml of 2xTYAG was added to theplates and the cells were scraped form the surface and transferred to a50 ml polypropylene tube. 2xTYAG containing 50% glycerol was added tothe cell suspension to obtain a final concentration of 17% glycerol.Aliquots of the selection round were kept at −80° C.

Phage Rescue.

100 μl of cell suspension obtained from previous selection rounds wereadded to 20 ml of 2xTYAG and grown at 37° C. with agitation (240 rpm)until an OD₆₀₀ of 0.3 to 0.5 was reached. The culture was thensuper-infected with 3.3×10¹⁰ MK13K07 helper phage and incubated for onehour at 37° C. (150 rpm). The medium was then changed by centrifugingthe cells at 2000 rpm for 10 minutes, removing the medium andresuspending the pellet in 20 ml of 2xTY-AK (100 μg/ml ampicillin; 50μg/ml kanamycin). The culture was then grown overnight at 30° C. (240rpm).

Monoclonal Phage Rescue for ELISA.

Single clones were picked into a microtiter plate containing 150 μl of2xTYAG media (2% glucose) per well and grown at 37° C. (100-120 rpm) for5-6 h. M13K07 helper phage was added to each well to obtain amultiplicity of infection (MOI) of 10 (i.e., 10 phage for each cell inthe culture) and incubated at 37° C. (100 rpm) for 1 h. Followinggrowth, plates were centrifuged at 3,200 rpm for 10 min. Supernatant wascarefully removed, cells re-suspended in 150 μl 2xTYAK medium and grownovernight at 30° C. (120 rpm). For the ELISA, the phage are blocked byadding 150 μl of 2× concentration PBS containing 5% skimmed milk powderfollowed by one hour incubation at room temperature. The plates werethen centrifuged 10 minutes at 3000 rpm and the phage containingsupernatant used for the ELISA.

Phage ELISA.

ELISA plates (Maxisorb, NUNC) were coated overnight with 2 μg/ml hIFNγin PBS. Control plates were coated with 2 μg/ml BSA. Plates were thenblocked with 3% skimmed milk/PBS at room temperature for 1 h. Plateswere washed 3 times with PBS 0.05% Tween 20 before transferring thepre-blocked phage supernatants and incubation for one hour at roomtemperature. Plates were then washed 3 times with PBS 0.05% Tween 20. 50μl of 3% skimmed milk/PBS containing (HRP)-conjugated anti-M13 antibody(Amersham, diluted 1:10,000) to each well. Following incubation at roomtemperature for 1 hr, the plates were washed 5 times with PBS 0.05%Tween 20. The ELISA was then revealed by adding 50 μl of TMB (Sigma) and50 μl of 2N H₂SO₄ to stop the reaction. Absorption intensity was read at450 nm.

Phage Clone Sequencing

Single clones were placed in a microtiter plate containing 150 μl of2xTYAG media (2% glucose) per well and grown at 30° C. (120 rpm)overnight. The next day 5 μl of culture was transferred into anotherplate containing 45 μl of dH₂O and mixed. The plates was then frozen at−20° C. After thawing, 1 μl of this suspension was used for PCRamplification using standard PCR protocols with primer specific forpCANTAB6: mycseq, 5′-CTCTTCTGAGATGAGTTTTTG-3′ (SEQ ID NO:100) andgene3leader, 5′-TTATTATTCGCAATTCCTTTAGTTGTTCCT-3′ (SEQ ID NO:101).

The PCR reactions were purified in 96 well format using the MontagePCRμ96 system (Millipore). 5 μl of the eluted DNA was sequencing usingthe mycseq and gene3leader primers.

ScFv Periplasmic Preparation for Functional Tests.

Individual clones were inoculated into a deep well microtiter platecontaining 0.9 ml of 2xTYAG media (0.1% glucose) per well and grown at37° C. for 5-6 h (250 rpm). 100 μl per well of 0.2 mM IPTG in 2xTYmedium were then added to give a final concentration of 0.02 mM IPTG.Plates were then incubated overnight at 30° C. with shaking at 250 rpm.The deep-well plates were centrifuged at 2,500 rpm for 10 min and thesupernatant carefully removed. The pellets were re-suspended in 150 μlTES buffer (50 mM Tris/HCl (pH 8), 1 mM EDTA (pH 8), 20% sucrose,complemented with Complete protease inhibitor, Roche). A hypotonic shockwas produced by adding 150 μl of diluted TES buffer (1:5 TES:waterdilution) and incubation on ice for 30 min. Plates were then centrifugedat 4000 rpm for 10 minutes to remove cells and debris. The supernatantswere carefully transferred into another microtiter plate and kept on icefor immediate testing in functional assays or ELISAs.

Large Scale scFv Purification

A starter culture of 1 ml of 2xTYAG was inoculated with a single colonyfrom a freshly streaked 2xTYAG agar plate and incubated with shaking(240 rpm) at 37° C. for 5 hours. 0.9 ml of this culture was used toinoculate a 400 ml culture of the same media and was grown overnight at30° C. with vigorous shaking (300 rpm).

The next day the culture was induced by adding 400 μl of 1M IPTG andincubation was continued for an additional 3 hours. The cells werecollected by centrifugation at 5,000 rpm for 10 minutes at 4° C.Pelleted cells were resuspended in 10 ml of ice-cold TES buffercomplemented with protease inhibitors as described above. Osmotic shockwas achieved by adding 15 ml of 1:5 diluted TES buffer and incubationfor 1 hour on ice. Cells were centrifuged at 10,000 rpm for 20 minutesat 4° C. to pellet cell debris. The supernatant was carefullytransferred to a fresh tube. Imidazole was added to the supernatant to afinal concentration of 10 mM. 1 ml of Ni-NTA resin (Qiagen),equilibrated in PBS was added to each tube and incubated on a rotarymixer at 4° C. (20 rpm) for 1 hour.

The tubes were centrifuged at 2,000 rpm for 5 minutes and thesupernatant carefully removed. The pelleted resin was resuspended in 10ml of cold (4° C.) Wash buffer 1 (50 mM NaH₂PO₄, 300 mM NaCl, 10 mMimidazole, pH to 8.0). The suspension was added to a polyprep column(Biorad). 8 ml of cold Wash Buffer 2 (50 mM NaH₂PO₄, 300 mM NaCl, 20 mMimidazole, pH to 8.0) were used to wash the column by gravity flow. ThescFv were eluted from the column with 2 ml of Elution buffer (50 mMNaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH to 8.0). Fractions wereanalyzed by absorption at 280 nm and protein containing fractions werepooled before buffer exchange on a PD10 desalting column (Amersham)equilibrated with PBS. The scFv in PBS were analyzed by SDS-PAGE andquantified by absorption at 280 nm. The purified scFv were aliquoted andstored at −20° C. and at 4° C.

Example 4: scFv Extract Inhibition of Interferon Gamma-Induced ReporterGene Expression

Periplasmic scFv extracts of various huIFNγ antibodies were produced asdescribed above. A high through-put screen cell-based assay was used forthe identification of single chain variable fragment (scFv) blockers ofIFNγ activity. A reporter gene (firefly luciferase), driven by theIFNγ-inducible GBP1 promoter, was transfected into the human melanomacell line, Me67.8. Both scFv and IFNγ were added to the cell cultureconcomitantly. Following a 6 hour incubation time, the luciferasereporter assay was performed. The scFv found to inhibit the induction offirefly luciferase were retained for further validation.

Several scFv extracts inhibited the IFNγ-induced reporter gene in a dosedependent fashion (FIG. 15). For each scFv clone shown in FIG. 15,various concentrations (2.7, 0.68, 0.17, 0.043 and 0.011 nM) were testedas shown by the columns above each clone name (descending concentrationfrom left to right). The percentage inhibition exhibited by each scFvextract at these various concentrations is shown in Table 3 below.

TABLE 3 Percentage inhibition of IFNγ-induced reporter gene expressionby periplasmic scFV extracts [scFv] nM A6 H8 A8 D8 C10 B4 B9 F9 A4 E1 C9G7 G10 D6 G6 G9 D3 F8 2.7 82 80 85 60 63 63 62 68 36 43 56 75 47 82 7352 69 64 0.68 92 75 86 60 50 49 53 32 1 68 67 73 44 73 66 12 55 64 0.17100 65 87 40 53 21 56 3 0 38 58 24 0 31 25 0 36 48 0.043 87 26 65 10 345 0 0 0 10 33 38 0 11 0 0 28 0 0.011 0 0 13 0 19 0 0 0 0 0 0 4 0 0 0 020 0

Example 5: scFv Inhibition of Interferon Gamma-Induced MHC Class IIExpression

A flow cytometric assay was implemented to identify fully human IgGantibodies, or fragments thereof, capable of blocking the expression ofIFNγ-induced MEW class II molecules. Following the plating of Me67.8cells, 5 ng/ml recombinant human IFNγ was added to cultures in thepresence of various concentrations of candidate fully human anti-IFNγmonoclonal antibodies. Following 48 h in culture, cells were stainedwith fluorescently labeled anti-human MEW class II antibody (HLA-DR) andanalyzed using a FACSCalibur®. Thus, the IC₅₀ (where 50% of theIFNγ-induced MHC class II expression is inhibited, i.e., 50% inhibitoryconcentration), for each candidate antibody is measured.

Purified fully human scFv were produced as described above in Example 1.The effect of the scFv on IFNγ-induced MHC class II expression onmelanoma cells was evaluated using the flow cytometric cell-based assaydescribed above. These scFv inhibited IFNγ-induced MHC II expression onmelanoma cells. (FIG. 16, Panels 1-12). The ability of these scFv clonesto inhibit IFNγ-induced MHC II expression on melanoma cells was comparedto a mouse anti-human IFNγ mAb referred to herein as 16C3. scFv clones(-) and the mouse anti-human IFNγ mAb 16C3 (---) are depicted in FIG.16.

Example 6: Reformatting scFv into IgG Format

Purified fully human scFv were produced as described above in Example 1.The V_(H) and V_(L) sequence of selected scFv were amplified withspecific oligonucleotides introducing a leader sequence and a HindIIIrestriction site at the 5′ end. An ApaI or an AvrII site was introducedat the 3′ end of the heavy and light chain sequence, respectively. Theamplified V_(H) sequences were digested HindIII/ApaI and cloned into thepCon_gamma1 expression vector (LONZA, Basel, Switzerland). The amplifiedV_(L) sequences were digested HindIII/AvrII and cloned into thepCon_lambda2 expression vector (LONZA). The constructions were verifiedby sequencing before transfection into mammalian cells.

The V_(H) and V_(L) cDNA sequences in their appropriate expressionvectors were transfected into mammalian cells using the Fugene 6Transfection Reagent (Roche, Basel, Switzerland). Briefly, Peak cellswere cultured in 6-well plates at a concentration of 6×10⁵ cells perwell in 2 ml culture media containing fetal bovine serum. The expressionvectors, encoding the candidate V_(H) and V_(L) sequences, wereco-transfected into the cells using the Fugene 6 Transfection Reagentaccording to manufacturer's instructions. One day followingtransfection, the culture media was aspirated, and 3 ml of freshserum-free media was added to cells and cultured for three days at 37°C. Following three days culture period, the supernatant was harvestedfor IgG purified on protein G columns.

Reformatted fully IgG was purified from serum-free supernatants fromtransfected cells using Protein G-Sepharose 4B fast flow columns (Sigma,St. Louis, Mo.) according to manufacturer's instructions. Briefly,supernatants from transfected cells were incubated overnight at 4° C.with ImmunoPure (G) IgG binding buffer (Pierce, Rockford Ill.). Sampleswere then passed over Protein G-Sepharose 4B fast flow columns and theIgG consequently purified using elution buffer. The eluted IgG fractionwas then dialyzed against PBS and the IgG content quantified byabsorption at 280 nm. Purity and IgG integrity were verified bySDS-PAGE.

Example 7: Inhibition of Interferon Gamma-Induced MHC Class IIExpression by Reformatted scFv

scFv were reformatted into an IgG format as described above. The effectof the IgG clones on IFNγ-induced MHC class II expression on melanomacells was evaluated using the flow cytometric cell-based assay describedabove in Example 2. As shown in FIG. 17, Panels 1-7, these IgG clonesinhibited IFNγ-induced MHC II expression on melanoma cells. The abilityof these IgG clones to inhibit IFNγ-induced MHC II expression onmelanoma cells was compared to the mouse anti-human IFNγ mAb 16C3 andthe R&D Systems mouse anti-human IFNγ antibody MAB285. Fully IgG clones(-x-), the mouse anti-human IFNγ mAb 16C3 (-▴-), and the R&D Systems,Inc. (Minneapolis, Minn.) mouse anti-human IFNγ MAB285 (--) aredepicted.

The IC₅₀ values for these IgG clones are shown below in Table 4.

TABLE 4 IC₅₀ analysis of fully human anti-IFNγ monoclonal antibodies.MHC II Inhibition IgG mAb Cell-Based Assay IC₅₀ 16C3 100 pM MAB285 400pM AC1.2R3P2_A6 41 pM AD14R4P1_B9 322 pM AC1.4R4P2_C10 203 pMAC1.2R3P2_D8 708 pM AD1.3R3P5_F8 1525 pM AD1.3R3P6_F9 185 pM AD14R4P2_G7233 pM

Example 8: Back-Mutation of huIFNγ Antibody Clone to Germline Sequence

In the studies described herein, the nucleotides and amino acid residuesin the nucleic acid and amino acid sequence of the A6 clone were mutatedto correspond to the nucleotide or amino acid residue found in thegermline sequence. This process is referred to herein as“back-mutation”.

A6 Heavy Chain:

The immunoglobulin heavy variable gene of antibody A6 had a 100%homology to the human germ line DP-47 or IGHV3-23 (GenBank Accessionnumber M99660). The immunoglobulin heavy joining (IGHJ) region of A6 wascompared to the six human functional IGHJ regions. The IGHJ region of A6was identified as IGHJ2 (Table 5A below), but had a better overallhomology with IGHJ5-02 (Table 5B below). The original IGHJ region of A6was therefore mutated to correspond to the sequence of IGHJ5-02, butonly for the sequence outside the CDR3. Mutated nucleotides and aminoacid residues are shown in boxes in Tables 5A and 5B, and the CDRregions are underlined.

TABLE 5A Comparison between A6 and human functional IGHJ2 genes

TABLE 5B Comparison between A6 and human functional IGHJ5-02 gene

A6 Light Chain:

The immunoglobulin lambda variable gene (VL) of antibody A6 belongs tothe IGLV6-57 or V1-22 subgroup (GenBank Accession Number Z73673). A6-VLhas 7 mutations compared to IGLV6-57, three in the CDRs and four in theframeworks (Table 6 below). The mutated nucleotides and amino acidresidues are shown in boxes in Table 6, and the CDR regions areunderlined.

The four mutations in framework regions are: Ser to Ala in framework 2region at Kabat position 43; Ser to Thr in framework 3 region at Kabatposition 72; Lys to Glu and Thr to Ala in framework 3 region at Kabatpositions 79 and 80, respectively. The four mutations in the frameworkregions were changed first individually, then all together back to thecorresponding human germ line residue. The mutations of these fourresidues back to the corresponding human germ line amino acid did notalter in any way the binding affinity of the NI-0501 antibody, alsoreferred to herein as “backmutated A6”, to its target antigen ascompared to the A6 antibody. The mutations from the A6 VL sequence tothe corresponding germ line residue in CDR1 (Ala to Val) and CDR2 (Glnto Arg) were carried out and were shown not to modify the overallaffinity for huIFNγ of the NI-0501 antibody (backmutated A6) as comparedto the A6 antibody.

TABLE 6 Comparison between A6 and human functional IGHV6-57 gene

The complete sequences of the NI-0501 heavy and light chains are setforth in FIGS. 1A-1D. The nucleotides and amino acid residues that werebackmutated to produce the NI-0501 antibody (i.e., those nucleotides andresidues that were changed from the original A6 sequence) are underlinedand italicized in FIGS. 1A and 1C.

Example 9: Affinity and Binding Kinetics of huIFNγ Antibody

The affinity and binding kinetics of the NI-0501 huIFNγ antibody werecharacterized on a Biacore 2000 instrument (Biacore AB, Uppsala,Sweden). 200 RU of NI-0501 were immobilized by EDC/NHS chemistry on a C1Biacore chip. Binding was measured by passing hIFNγ (R&D Systems) inHBS-EP buffer at concentrations between 200 nM and 1 nM. The flow ratewas 100 μl/minute and the temperature set at 25° C. The data was fittedaccording to 1:1 Langmuir model and the K_(on), K_(off) and K_(D) valuesdetermined (FIG. 18).

Example 10: Activity of huIFNγ Antibody

The activity of the NI-0501 huIFNγ antibody was compared to the activityof the antibody produced by the clone A6 (i.e., the A6 huIFNγ antibody).In this study, the ability of each huIFNγ antibody to inhibitrecombinant human IFNγ (rhuIFNγ)-induced MHC class II upregulation onthe human melanoma cell line, Me67.8 was evaluated. Briefly, Me67.8melanoma cells were incubated with rhuIFNγ, in the presence of NI-0501or the A6 huIFNγ antibody for 48-72 h. MHC class II upregulation wasmeasured as described above in Example 5. The two antibodies presented asimilar activity, which demonstrates that the backmutations in theNI-0501 huIFNγ antibody did not modify the activity of the antibody(FIG. 19).

The activity of the NI-0501 huIFNγ antibody was then tested on nativeIFNγ. In this study, human peripheral blood mononuclear cells (PBMCs)were activated with 1 μg/ml of the mitogen PHA for 48 h, andsupernatants were tested via ELISA for the presence of native IFNγ. Thissupernatant was then used to stimulate the MHC class II upregulation onMe67.8 cells. NI-0501 was able to neutralize the MHC class IIupregulation induced by native human IFNγ (FIG. 20).

Example 11: Cross-Reactivity of huIFNγ Antibody

Binding Assay:

NI-0501 was tested for its ability to bind to IFNγ using a SandwichELISA format assay. Briefly, IFNγ from the species mentioned in thetitle of each graph shown in FIG. 21 was captured with pre-coatedNI-0501 (-▴-) or the control anti-species IFNγ mAb (-▪-). The IFNγ fromeach species was detected using a polyclonal antibody specific for theIFNγ in that assay. As seen in FIG. 21, NI-0501 binding to rat IFNγ issimilar to the control antibody, but not for the other species,excluding cynomolgus monkey.

Neutralization of IFNγ Activity:

The antibody NI-0501 was tested for its ability to neutralize or inhibitrecombinant IFNγ proteins from several different species. Briefly,recombinant IFNγ from the various tested species was placed in culturewith Me67.8 cells in the presence or absence of NI-0501 for 48-72 h. MHCclass II upregulation was measured as described above in Example 5. Thecross-reactivity to, and neutralization of, cynomolgus IFNγ wasdemonstrated by inhibiting the MHC class II upregulation on the humanmelanoma cell line, Me67.8 (FIG. 22). NI-0501 was able to inhibit IFNγfrom cynomolgus monkey but could not neutralize IFNγ from the othertested species, demonstrating no cross reactivity the antibody withthese species (Table 7).

TABLE 7 Cross Reactivity of NI-0501 NI-0501 nhuIFNγ rhuIFNγ ncyIFNγrcyIFNγ rdIFNγ rcIFNγ rrIFNγ rmIFNγ Binding + + + + − − − −Neutralization + + + + * * * * nhu = native human IFNγ rhu = recombinanthuman IFNγ ncy = native cynomolgus IFNγ rcy = recombinant cynomolgusIFNγ rd = recombinant dog IFNγ rc = recombinant cat IFNγ rr =recombinant rat IFNγ rm = recombinant mouse IFNγ + = cross-react − = donot cross-react

In addition, cynomolgus PBMCs were activated with 1 μg/ml of the mitogenPHA for 48 h, and supernatants were tested via ELISA for the presence ofnative IFNγ. This supernatant was then used to stimulate the MHC classII upregulation on Me67.8. NI-0501 was able to neutralize the MHC classII upregulation induced by native cynomolgus IFNγ (FIG. 22). *=nottested

Example 12: Biological Activity of huIFNγ Antibody

The studies described herein were designed to test the biologicalactivity of the NI-0501 huIFNγ antibody upon administration tocynomolgus monkeys. NI-0501 was chosen for the safety andpharmacokinetics (PK) studies described herein because this huIFNγantibody was found to cross-react with the IFNγ from cynomolgus monkeys,as described above. To evaluate adverse clinical effects after multipleintravenous infusions, monkeys are infused with the following doses: 30mg/kg, 100 mg/kg and 200 mg/kg.

In mice with a disrupted IFNγ gene, decreased levels of IgG2a andincreased levels of IgG1 were observed in response to KLH immunization,demonstrating the correlation between IFNγ and the IgG response. Duringthe 13 week main toxicology study, monkeys are immunized with KLH inIncomplete Freund's Adjuvant (IFA). A typical immune response to KLH/IFAin monkeys, co-treated with placebo, elicits a KLH-specific IgM and IgGresponse detectable in the serum. These studies are designed to evaluatewhether neutralizing IFNγ in NI-0501-treated monkeys that were immunizedwith KLH in IFA, alters the KLH-specific IgG titer.

Example 13: Modulation of IFNγ Activity Using huIFNγ Antibodies

The production of the chemokine IP-10 is up-regulated by IFNγ in severaldifferent cell lines. Based on this observation a whole blood assay wasdeveloped. In this whole blood assay, whole blood samples from severaldonors were mixed with a fixed concentration of IFNγ and differentconcentrations of NI-0501. After incubation, IP-10 levels were measuredby ELISA as a means for evaluating the efficacy of the anti-IFNγantibody to block the production of IP-10 (FIG. 23).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated fully human monoclonal anti-IFNγantibody or fragment thereof, wherein said antibody comprises: (a) aV_(H) CDR1 region comprising the amino acid sequence SYAMS (SEQ ID NO:3)or SNAMS (SEQ ID NO:43); (b) a V_(H) CDR2 region comprising the aminoacid sequence AISGSGGSTYYADSVKG (SEQ ID NO:4) or TLTGSGGTAYYADSVEG (SEQID NO:44), and (c) a V_(H) CDR3 region comprising an amino acid sequenceselected from the group consisting of DGSSGWYVPHWFDP (SEQ ID NO:5);DHSSGWYVISGMDV (SEQ ID NO:13); DLTVGGPWYYFDY (SEQ ID NO:21); DGWNALGWLES(SEQ ID NO:29); GTELVGGGLDN (SEQ ID NO:45); RSFDSGGSFEY (SEQ ID NO:64);VGSWYLEDFDI (SEQ ID NO:69); GGNYGDYFDYFDY (SEQ ID NO:76); andDFWVITSGNDY (SEQ ID NO:89), (d) a V_(L) CDR1 region comprising an aminoacid sequence selected from the group consisting of TRSSGSIASNYVQ (SEQID NO:8); TRSSGSIASNYVQ (SEQ ID NO:16); TRSGGSIGSYYVQ (SEQ ID NO:32);TRSSGTIASNYVQ (SEQ ID NO:39); TGSGGSIATNYVQ (SEQ ID NO:48);TGSSGSIASNYVQ (SEQ ID NO:55); TRSSGSIASNYVH (SEQ ID NO:72);TGRNGNIASNYVQ (SEQ ID NO:84); AGSSGSIASNYVQ (SEQ ID NO:97) andTRSSGSIVSNYVQ (SEQ ID NO:106); (e) a V_(L) CDR2 region comprising anamino acid sequence selected from the group consisting of EDNQRPS (SEQID NO:9); EDNQRPS (SEQ ID NO:17); DDDQRPS (SEQ ID NO:25); DDKKRPS (SEQID NO:33); EDTQRPS (SEQ ID NO:85) and EDNRRPS (SEQ ID NO:107); and (f) aV_(L) CDR3 region comprising an amino acid sequence selected from thegroup consisting of QSYDGSNRWM (SEQ ID NO:10); QSNDSDNVV (SEQ ID NO:18);QSYDSSNVV (SEQ ID NO:26); QSYDSNNLVV (SEQ ID NO:34); QSYDNSNHWV (SEQ IDNO:40); QSYDSDNHHVV (SEQ ID NO:49); QSYDSSNQEVV (SEQ ID NO:56);QSYDSNNFWV (SEQ ID NO:61); QSSDTTYHGGVV (SEQ ID NO:73); QSYEGF (SEQ IDNO:79); QSSDSNRVL (SEQ ID NO:86); QSFDSTNLVV (SEQ ID NO:92); andQSYSYNNQVV (SEQ ID NO:98), wherein said antibody binds IFNγ.
 2. Theantibody of claim 1, wherein said antibody is an IgG isotype.
 3. Theantibody of claim 1, wherein said antibody comprises a V_(H) CDR1 regioncomprising the amino acid sequence SYAMS (SEQ ID NO:3); a V_(H) CDR2region comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ IDNO:4), a V_(H) CDR3 region comprising the amino acid sequenceDGSSGWYVPHWFDP (SEQ ID NO:5); a V_(L) CDR1 region comprising the aminoacid sequence TRSSGSIASNYVQ (SEQ ID NO:8); a V_(L) CDR2 regioncomprising the amino acid sequence EDNQRPS (SEQ ID NO:9); and a V_(L)CDR3 region comprising an amino acid sequence QSYDGSNRWM (SEQ ID NO:10).4. An isolated fully human monoclonal antibody, wherein said antibodycomprises a heavy chain variable amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2, 12, 20, 28, 36, 42, 51, 58, 63, 68,75, 81, 88, 94 or 103, and a light chain variable amino acid sequenceselected from the group consisting of SEQ ID NOS: 7, 15, 23, 31, 38, 47,54, 60, 66, 71, 78, 83, 91, 96 or 105, wherein said antibody binds IFNγ.5. The antibody of claim 4, wherein said antibody is an IgG isotype. 6.A pharmaceutical composition comprising an antibody of claim 1 and acarrier.
 7. A pharmaceutical composition comprising an antibody of claim4 and a carrier.
 8. A method of alleviating a symptom of an autoimmunedisease or inflammatory disorder, the method comprising administering anantibody of claim 1 to a subject in need thereof in an amount sufficientto alleviate the symptom of the autoimmune disease or inflammatorydisorder in the subject.
 9. The method of claim 8, wherein said subjectis a human.
 10. The method of claim 8, wherein said antibody comprises aV_(H) CDR1 region comprising the amino acid sequence SYAMS (SEQ IDNO:3); a V_(H) CDR2 region comprising the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO:4), a V_(H) CDR3 region comprising theamino acid sequence DGSSGWYVPHWFDP (SEQ ID NO:5); a V_(L) CDR1 regioncomprising the amino acid sequence TRSSGSIASNYVQ (SEQ ID NO:8); a V_(L)CDR2 region comprising the amino acid sequence EDNQRPS (SEQ ID NO:9);and a V_(L) CDR3 region comprising an amino acid sequence QSYDGSNRWM(SEQ ID NO:10).
 11. The method of claim 8, wherein said autoimmunedisease or inflammatory disorder is selected from the group consistingof Crohn's Disease, systemic lupus erythematosus, psoriasis, rheumatoidarthritis, vasculitis, atopic dermatitis and secondary progressivemultiple sclerosis.
 12. The method of claim 8, wherein said antibody isadministered intravenously.
 13. The method of claim 8, wherein saidantibody is co-administered with an second agent selected from the groupconsisting of: (a) an anti-cytokine that recognizes one or morecytokines selected from interleukin 1 (IL-1), IL-2, IL-4, IL-6, IL-12,IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27 and IL-31;(b) an anti-chemokine reagent that recognizes one or more cytokinesselected from IL-1, IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18,IL-20, IL-21, IL-22, IL-23, IL-27 and IL-31; (c) a chemokines selectedfrom MIP1 alpha, MIP1 beta, RANTES, MCP1, IP-10, ITAC, MIG, SDF andfractalkine.
 14. A method of reducing MHC class II expression on a cell,the method comprising contacting a cell with an antibody of claim 1 inan amount sufficient to reduce MHC class II expression on said cell. 15.The method of claim 14, wherein said cell is a human melanoma cell. 16.The method of claim 14, wherein said antibody comprises a V_(H) CDR1region comprising the amino acid sequence SYAMS (SEQ ID NO:3); a V_(H)CDR2 region comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ IDNO:4), a V_(H) CDR3 region comprising the amino acid sequenceDGSSGWYVPHWFDP (SEQ ID NO:5); a V_(L) CDR1 region comprising the aminoacid sequence TRSSGSIASNYVQ (SEQ ID NO:8); a V_(L) CDR2 regioncomprising the amino acid sequence EDNQRPS (SEQ ID NO:9); and a V_(L)CDR3 region comprising an amino acid sequence QSYDGSNRWM (SEQ ID NO:10).17. The method of claim 14, wherein said cell is contacted with a secondagent selected from the group consisting of: (a) an anti-cytokine thatrecognizes one or more cytokines selected from interleukin 1 (IL-1),IL-2, IL-4, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21,IL-22, IL-23, IL-27 and IL-31; (b) an anti-chemokine reagent thatrecognizes one or more cytokines selected from IL-1, IL-2, IL-4, IL-6,IL-12, IL-13, IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-27 andIL-31; (c) a chemokines selected from MIP1 alpha, MIP1 beta, RANTES,MCP1, IP-10, ITAC, MIG, SDF and fractalkine.